| 4Background:Liver cancer ranks sixth in cancer incidence and second in cancer mortality in worldwide,with more than half of newly diagnosed cases and deaths occur in China.With difficulties of early diagnosis and rapid progression,the survival rate of liver cancer is very low.Surgery and liver transplantation are the best treatment for patients with liver cancer,but the clinical treatment of unresectable liver cancer is still a challenge.Recently,a clinical trial has demonstrated the combination of transarterial chemoembolisation（TACE）plus sorafenib can significantly improve the progression-free survival in patients with unresectable liver cancer.However,TACE is a locoregional treatment approach that is not appropriate for advanced or metastatic liver cancer.Objective:In this study,we proposed a salined CA4 nanoparticles（CA4-NPs）at the base of poly（L-glutamic acid）-graft-methoxy poly（ethylene glycol）/combretastatin A4.CA4-NPs can disrupt the established tumor vasculature and result in tumor vessel occlusion after administration,which is similar to TACE in reducing tumor blood-flow.And the CA4-NPs treatment has a comparative advantage of systemic therapy.In this paper,we discuss the anticancer efficiency,safety and simple mechnism of CA4-NPs plus sorafenib for the treatment of liver cancer by using H22tumor-bearing mouse models.Methods:CA4-NPs was prepared and be easily modified.H22 subcutaneous hepatoma transplantation model was constructed,and the mice were randomly divided into four group（PBS,CA4-NPs,sorafenib,sorafenib+CA4-NPs）.To evaluate the antitumor effect of each treatment group,we monitored the tumor volume,survival time,HE staining and Ki-67 immunohistochemistry（IHC）.Drug toxicity was assessed by monitoring the mice body weight change,serum liver and kidney function,and pathological results of major organs.CD31 IHC analysis was evaluated to identify the density of tumor blood vessels.The possible mechnism was determined by detecting the expression of VEGf-A.Finally,H22 orthotopic hepatoma transplantation model was established to verify the therapeutic effect of CA4-NPs combined with sorafenib in orthotopic liver cancer models.Results:1.CA4-NPs were successfully prepared.The drug characterization results were as follows:DLC%:20.0%,free CA4 content:2.0%;The average hydrodynamic radius of CA4-NPs measured by DLS was 42.9 nm.2.When the drug dose of CA4-NPs was 20 mg·kg-1（on the CA4 basis）,tumor inhibition results against H22 substaneous hepatoma model were as follows:（1）Tumor volumes or tumor inhibition rates:compared with the PBS group,both the CA4-NPs group and the sorafenib group could significantly inhibit tumor growth,and the combination group achieved the best antitumor effect.On day 14,the tumor suppression rates（TSR%）of mice in CA4-NPs(20 mg·kg-1 on the CA4 basis)group and sorafenib(30 mg·kg-1)group are 33.0%and 54.7%.TSR%in the combination treatment group was 81.6%,which was significantly higher than those of CA4-NPs and sorafenib group.（2）Survival time:the median survival time of 4 groups were 23days,24 days,29 days and 42 days,respectively.CA4-NPs alone with a dose of 20mg·kg-1 on the CA4 basis did not significantly prolong the survival period of mice over PBS group.However,both sorafenib monotherapy and the combination treatment yielded a remarkable survival advantage compared with PBS group.（3）HE images determines the tumor necrosis area:the necrotic area of CA4-NPs(20 mg·kg-1on the CA4 basis)group and sorafenib sorafenib(30 mg·kg-1)group were（46.8±3.3）%and（44.6±0.5）%,respectively.Whereas,this increased to（86.9±5.6）%in the combination therapy group.（4）Ki-67 IHC:the highest expression of Ki-67 in the PBS group of H22 tumor-bearing mice,while the least expression of Ki-67 was present in the combination group.3.When the drug dose of CA4-NPs was 20 mg·kg-1（on the CA4 basis）,the drug toxicity results on H22 subcutaneous hepatoma model were as follows:（1）Body weight change rate:weight loss was not more than 20%in all treatment groups.（2）Liver and kidney function:the serum AST levels were significantly decreased in the three treatment groups（CA4-NPs,sorafenib,sorafenib+CA4-NPs）compared with the PBS group（P<0.05）.There was no statistical difference in the serum levels of ALT and BUN among all groups（P>0.05）.（3）HE staining:There was no obvious tissue injury of major organs at the end of the treatment.4.CD31 IHC results:（1）The average MVD of CD31 positive sections was remarkable lower on day 2 and day 7 compared with that on day 0 after CA4-NPs administration.However,MVD was apparently higher on day 14（73.0±4.6）,almost2 times of that on day 0.（2）On day 14 after treatment,compared with PBS group,MVD was significantly higher in CA4-NPs group than PBS group,decreased in sorafenib group,and the sorafenib+CA4-NPs group showed the minimum average MVD.5.VEGF-A protein expression:On day 14 after treatment,compared with PBS group,VEGF-A was upregulated in CA4-NPs group;sorafenib can significantly inhibit the expression of VEGF-A,and shows the minimum expression of VEGF-A.VEGF-A was significantlyless expressed after the application of sorafenib in conjunction with CA4-NPs compared with the CA4-NPs group.6.We monitored the tumor inhibition rates,body weight change rates and survival time of subcutaneous H22 tumor-bearing mice treated with CA4-NPs（30mg·kg-11 on the CA4 basis）,CA4-NPs（35 mg·kg-11 on the CA4 basis）,sorafenib(30mg·kg-1)+CA4-NPs(30 mg·kg-1 on the CA4 basis)or sorafenib(30 mg·kg-1)+CA4-NPs(35 mg·kg-1on the CA4 basis).（1）Tumor inhibition rate:all these four treatment therapies efficiently reduce the tumor burden.On day 14,the TSR%of mice receiving CA4-NPs（30 mg·kg-11 on the CA4 basis）or CA4-NPs（35 mg·kg-11 on the CA4 basis）was 51.7%or 95.3%,respectively.As for the combination therapy groups,the TSR%of mice was 92.9%for sorafenib(30 mg·kg-1)+CA4-NPs（30 mg·kg-11 on the CA4 basis）group,and 93.7%for sorafenib(30 mg·kg-1)+CA4-NPs(35 mg·kg-1on the CA4 basis)group.（2）Tumor body weight change rate:weight loss was not more than 20%and no mice died during the treatment period in all treatment groups.（3）Survial time:the median survival time of mice receiving CA4-NPs(30 mg·kg-1 on the CA4 basis)was 24 days,and 40 days for the CA4-NPs(35 mg·kg-1 on the CA4basis)treatment group.After 90 days of first treatment,the median survival time did not reached,and there were 71.4%percent of mice were alive without residual tumor in the two combination therapy groups.7.The H22 orthotopic hepatoma model was successfully constructed to further verify the therapeutic effect of CA4-NPs combined with sorafenib.The results were as follows:compared with the PBS group,both CA4-NPs and sorafenib could significantly inhibit tumor growth,and tumor weight was lower than that of the PBS group.The combination therapy group showed the lightest tumor weight.HE staining indicated that the order of areas of tumor necrosis was calculated as follows:sorafenib+CA4-NPs>CA4-NPs or sorafenib>PBS.Conclusion:1.CA4 nanoparticles with high solubility was prepared successfully.2.The in vivo experiments of H22 subcutaneous hepatoma model confirmed that CA4-NPs combined with sorafenib can inhibit tumor growth and prolong survival time of H22 tumor-bearing mice.3.There were no obvious tissue injuries of major organs after being administrated with CA4-NPs and/or sorafenib.4.The MVD can be rapidly reduced and maintained for at least one week after being treated with CA4-NPs,but increased two weeeks later.After two weeks of treatment,the combination group exhibited the minimum amount of MVD.5.CA4-NPs could improve the expression of VEGF-A.Continuous adminstration of sorafenib could inhibit the high expression of VEGF-A induced by CA4-NPs,which resulting in lower expression of VEGF-A in the combination group compared with PBS and CA4-NPs group.6.CA4-NPs plus sorafenib treatment also played an effective anti-tumor role in the orthotopic hepatoma model. |
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